The Streptomyces phage BT1 encodes a site-specific integrase of the large serine recombinase subfamily. In this report, the enzymatic activity of the BT1 integrase was characterized in vitro. We showed that this integrase has efficient integration activity with substrate DNAs containing attB and attP sites, independent of DNA supercoiling or cofactors. Both intra-and intermolecular recombinations proceed with rapid kinetics. The recombination is highly specific, and no reactions are observed between pairs of sites including attB and attL, attB and attR, attP and attL, or attP and attR or between two identical att sequences; however, a low but significant frequency of excision recombination between attL and attR is observed in the presence of the BT1 integrase alone. In addition, for efficient integration, the minimal sizes of attB and attP are 36 bp and 48 bp, respectively. This site-specific recombination system is efficient and simple to use; thus, it could have applications for the manipulation of DNA in vitro.Site-specific recombinases have been widely used in genetic engineering: for example, in vitro cloning, plant and mammalian cell genome modification, and gene therapy (3,11,12,15,16). Nearly all site-specific recombinases can be classified as tyrosine recombinases, also known as the integrase family, or serine recombinases, also known as the resolvase/invertase family, based on comparisons of amino acid sequences and different mechanisms of catalysis; these two types use tyrosine or serine, respectively, to attack the DNA sugar-phosphate backbone (14,18,20). The process of strand exchange catalyzed by tyrosine recombinases involves a Holliday junction intermediate and cleavage and rejoining of the strands one by one. In contrast, serine-mediated recombination involves a process of double-strand breakage, followed by rotation and religation (9). The well-known tyrosine recombinase integrase recognizes two different attachment substrate sites, the 25-bp attB gene (in the bacterial chromosome) and the 240-bp attP gene (in the phage genome), which are inverted into attL and attR with the aid of the integration host factor (a chaperone). Furthermore, excision between attL and attR requires recombination directionality factor (RDF), and recombinations mediated by tyrosine recombinases require supercoiling of the substrate DNAs (10). In contrast, the extensively studied large serine recombinase from Streptomyces phage C31 is thought to catalyze unidirectional recombination between DNA substrates attB and attP, independent of DNA supercoiling and host cofactors (18). In addition, the attachment sites of the C31 integrase are smaller than those of most tyrosine recombinases, with minimal sizes of 34 bp for attB and 39 bp for attP (11,18).Streptomyces phage BT1, a homoimmune relative of C31, is inserted into the chromosomes of a wide range of Streptomyces spp. for lysogeny and encodes a large serine recombinase (5, 8). Similar to those of the C31 site-specific recombination system, the attB and attP sites of BT1 ...

The multicopper enzyme nitrous oxide reductase reduces the greenhouse gas N2O to uncritical N2as the final step of bacterial denitrification. Its two metal centers require an elaborate assembly machinery that so far has precluded heterologous production as a prerequisite for bioremediatory applications in agriculture and wastewater treatment. Here, we report on the production of active holoenzyme inEscherichia coliusing a two-plasmid system to produce the entire biosynthetic machinery as well as the structural gene for the enzyme. Using this recombinant system to probe the role of individual maturation factors, we find that the ABC transporter NosFY and the accessory NosD protein are essential for the formation of the [4Cu:2S] site CuZ, but not the electron transfer site CuA. Depending on source organism, the heterologous hostE. colican, in some cases, compensate for the lack of the Cu chaperone NosL, while in others this protein is strictly required, underlining the case for designing a recombinant system to be entirely self-contained.

DNA methylation is an epigenetic event involved in a variety array of processes that may be the foundation of genetic phenomena and diseases. DNA methyltransferase is a key enzyme for cytosine methylation in DNA, and can be divided into two functional families (Dnmt1 and Dnmt3) in mammals. All mammalian DNA methyltransferases are encoded by their own single gene, and consisted of catalytic and regulatory regions (except Dnmt2). Via interactions between functional domains in the regulatory or catalytic regions and other adaptors or cofactors, DNA methyltransferases can be localized at selective areas (specific DNA/nucleotide sequence) and linked to specific chromosome status (euchromatin/heterochromatin, various histone modification status). With assistance from UHRF1 and Dnmt3L or other factors in Dnmt1 and Dnmt3a/ Dnmt3b, mammalian DNA methyltransferases can be recruited, and then specifically bind to hemimethylated and unmethylated double-stranded DNA sequence to maintain and de novo setup patterns for DNA methylation. Complicated enzymatic steps catalyzed by DNA methyltransferases include methyl group transferred from cofactor Ado-Met to C5 position of the flipped-out cytosine in targeted DNA duplex. In the light of the fact that different DNA methyltransferases are divergent in both structures and functions, and use unique reprogrammed or distorted routines in development of diseases, design of new drugs targeting specific mammalian DNA methyltransferases or their adaptors in the control of key steps in either maintenance or de novo DNA methylation processes will contribute to individually treating diseases related to DNA methyltransferases.

The copper-containing enzyme nitrous oxide reductase (NOR) catalyzes the transformation of nitrous oxide (NO) to dinitrogen (N) in microbial denitrification. Several accessory factors are essential for assembling the two copper sites Cu and Cu, and for maintaining the activity. In particular, the deletion of either the transmembrane iron-sulfur flavoprotein NosR or the periplasmic protein NosX, a member of the ApbE family, abolishes NO respiration. Here we demonstrate through biochemical and structural studies that the ApbE protein from Pseudomonas stutzeri, where the nosX gene is absent, is a monomeric FAD-binding protein that can serve as the flavin donor for NosR maturation via covalent flavinylation of a threonine residue. The flavin transfer reaction proceeds both in vivo and in vitro to generate post-translationally modified NosR with covalently bound FMN. Only FAD can act as substrate and the reaction requires a divalent cation, preferably Mg that was also present in the crystal structure. In addition, the reaction is species-specific to a certain extent.

We describe a site-specific recombination-based tandem assembly (SSRTA) method for reconstruction of biological parts in synthetic biology. The system was catalyzed by Streptomyces phage φBT1 integrase, which belongs to the large serine recombinase subfamily. This one-step approach was efficient and accurate, and able to join multiple DNA molecules in vitro in a defined order. Thus, it could have applications in constructing metabolic pathways and genetic networks.